Deformability of Heterogeneous Red Blood Cells in Aging and Related Pathologies.

Aging is interrelated with changes in red blood cell parameters and functionality. In this article, we focus on red blood cells (RBCs) and provide a review of the known changes associated with the characterization of RBC deformability in aging and related pathologies. The biophysical parameters complement the commonly used biochemical parameters and may contribute to a better understanding of the aging process. The power of the deformability measurement approach is well established in clinical settings. Measuring RBCs' deformability has the advantage of relative simplicity, and it reflects the complex effects developing in erythrocytes during aging. However, aging and related pathological conditions also promote heterogeneity of RBC features and have a certain impact on the variance in erythrocyte cell properties. The possible applications of deformability as an early biophysical biomarker of pathological states are discussed, and modulating PIEZO1 as a therapeutic target is suggested. The changes in RBCs' shape can serve as a proxy for deformability evaluation, leveraging single-cell analysis with imaging flow cytometry and artificial intelligence algorithms. The characterization of biophysical parameters of RBCs is in progress in humans and will provide a better understanding of the complex dynamics of aging.

Systematic review and meta-analysis of cryopreserved bovine sperm assessment: harnessing imaging flow cytometry for multi-parametric analysis.

Cryopreservation of sperm is an essential technique in assisted reproduction in cattle. The objective of the study was to systematically review and synthesize the literature on bull semen quality evaluation based on the comparison of morphological and metabolic parameters of cryopreserved bovine spermatozoa such as DNA integrity, mitochondrial status, plasma membrane alterations, total motility, and morphology (% of abnormal cells). The electronic databases PubMed, Web of Sciences, Scopus, and Google Scholar were searched up to December 2023. Studies and references were included if they reported the following parameters: DNA integrity, mitochondrial status, plasma membrane alterations, total motility, and morphological aberrations (% of abnormal cells) for conventional cryopreserved bovine spermatozoa. After an electronic search, out of 1,526 original studies, only 40 were included in the meta-analysis. Standardized mean differences (SMD) with 95% confidence intervals were estimated for the chosen studies, and a meta-analysis was performed using a random effects model. The tau-squared (tau2) and inconsistency index (I2) quantified heterogeneity among different studies. The regression analysis for the evaluated parameters showed a positive correlation between mitochondrial membrane potential (MMP), total motility, and abnormal morphology and a negative correlation between DNA fragmentation index (DFI) and total motility and MMP. Moreover, subgroup analysis demonstrated similar associations for dairy and non-dairy bull breeds, albeit with lower I2 values. The presence of publication bias was confirmed by Egger's test, except for the MMP parameter. A multi-parametric analysis of morphological and metabolic parameters can address the existing limitations of cryopreserved bovine spermatozoa quality assessment. Combining imaging flow cytometry (IFC) with standardization of sperm pre-processing and optimization of the experimental protocols may help to differentiate sperm from cellular debris and cytoplasmic droplets of similar size and alleviate limitations demonstrated by conventional sperm analysis.

Effect of Focal Adhesion Kinase and Vinculin Expression on Migration Parameters of Normal and Tumor Epitheliocytes.

Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma.

In vitro and in vivo toxicity of thiolated and PEGylated organosilica nanoparticles.

This study comprises the comprehensive toxicological assessment of thiolated organosilica nanoparticles (NPs) synthesised from 3-mercaptopropyltrimethoxysilane (MPTS). We investigated the influence of three different types of nanoparticles synthesised from 3-mercaptopropyltrimethoxysilane: the starting thiolated silica (Si-NP-SH) and their derivatives prepared by surface PEGylation with PEG 750 (Si-NP-PEG750) and 5000 Da (Si-NP-PEG5000) on biological subjects from in vitro to in vivo experiments to explore the possible applications of those nanoparticles in biomedical research. As a result of this study, we generated a comprehensive understanding of the toxicological properties of these nanoparticles, including their cytotoxicity in different cell lines, hemolytic properties, in vitro localisation, mucosal irritation properties and biodistribution in BALB/c mice. Our findings indicate that all three types of nanoparticles can be considered safe and have promising prospects for use in biomedical applications. Nanoparticles did not affect the viability of HPF, MCF7, HEK293 and A549 cell lines at low concentrations (up to 100 µg/mL); moreover, they did not cause organ damage to BALB/c mice at concentrations of 10 mg/kg. The outcomes of this study enhance our understanding of the impact of organosilica nanoparticles on health and the environment, which is vital for developing silica nanoparticle-based drug delivery systems and provides opportunities to expand the applications of organosilica nanoparticles.